Method for culturing hcv virus in vitro

ABSTRACT

The invention concerns a method for HVC virus culture in vitro comprising steps which consist in: contacting particles containing hepatitis C virus RNA with cells capable of synthesizing and secreting lipoproteins, in a suitable culture environment promoting synthesis and secretion of lipoproteins, and in collecting the virus thus obtained. The invention has diagnostic and therapeutic applications.

The present invention relates to a novel method for culturing thehepatitis C virus in vitro.

Hepatitis C is the main cause of hepatitis acquired by transfusion.Hepatitis C can also be transmitted via other percutaneous routes, forexample by intravenous injection of drugs. The risk of healthprofessionals being contaminated is not, moreover negligible.

Hepatitis C differs from the other forms of virus-related liverdiseases, such as hepatitis A, B or D. Infections with the hepatitis Cvirus (HCV) are often chronic, resulting in liver diseases such ashepatitis, cirrhosis and carcinoma in a large number of cases.

Although the risk of transmission of the virus by transfusion hasdecreased due to the selection of blood donors, the frequency ofhepatitis C cases remains high. Currently, approximately 170 millionpeople throughout the world are chronically infected with HCV. The highrisk populations are found mainly among individuals who have undergone ablood transfusion and intravenous drug users, but asymptomatic blooddonors exist who do not belong to these high risk groups and in whomcirculating anti-HCV antibodies have been found. For the latter, theroute of infection has still not been identified.

HCV was the first hepatotropic virus isolated by means of molecularbiology techniques. The sequences of the virus genome were clonedwithout the viral particles having been visualized.

Currently, the only natural viral particles characterized in the bloodof infected patients are nonenveloped capsids, even though it is knownthat other forms of particles containing the viral RNA exist.

For convenience, the term “virus” will be used in the presentapplication to refer to any particle containing RNA of the HCV virus.The two terms will also be used without distinction.

HCV is a positive single-stranded RNA virus of approximately 9.5 kbwhich replicates by means of a copy of complementary RNA and the productof translation of which is a single polyprotein of approximately 3000amino acids. The 5′ end of the HCV genome corresponds to an untranslatedregion adjacent to the genes which encode the structural proteins, thecore protein of the nucleocapsid and the two envelope glycoproteins, E1and E2. The 5′ untranslated region and the core gene are relatively wellconserved in the various genotypes, but the E2 envelope proteins areencoded by a hypervariable region that is different from one isolate toanother. The 3′ end of the HCV genome contains the genes which encodethe nonstructural (NS) proteins and a well conserved 3′ noncodingregion.

Because of its genomic organization and its presumed mode ofreplication, HCV has been classified in a new genus of the Flaviviridaefamily, the hepaciviruses.

The term “HCV virus” refers to any virus species, among which are thestrains pathogenic for humans, the attenuated strains and the defectivestrains derived from said strains. Indeed, it is known that RNA virusesexhibit a high spontaneous mutation rate. Multiple strains can thereforeexist, which may be more or less virulent. It is within the scope ofthose skilled in the art to identify such strains, for example bynucleic acid and/or peptide sequence homology with respect to areference strain and/or by identifying a strain or an isolate withrespect to morphological and/or immunological criteria.

Many techniques have been developed for diagnosing an HCV inspection.For example, diagnostic immunoassays have been carried out for detectingantibodies directed against HCV proteins in the sera of patients. Thesynthesis of cDNA by reverse transcription of the viral RNA andamplification by PCR have also been used to detect the HCV genome, suchas indirect measurement of a potentially infectious virus in the sera ofchronically infected humans or those of experimentally infectedchimpanzees. Moreover, on the basis of gene cloning, hybridizationtechniques with a DNA probe have also been developed.

However, it is recognized that the existing diagnostic techniques lacksensitivity and/or specificity and/or suffer from difficulties ofimplementation. By way of example, with the probe hybridization method,it is impossible to distinguish between a virus with low infectiouscapacity and a virus with high infectious capacity. It is thereforenecessary, to inoculate a chimpanzee with the virus which must betested, and to test the resulting infection on the animal, but this isdifficult to carry out.

It is therefore of foremost importance, from a public health point ofview, to be able to develop specific, sensitive and practical methodsfor identifying and screening for HCV carriers. One of the solutionscould be to produce an effective system for culturing HCV in vitro,which would make it possible to obtain propagation of the virus, inparticular for studying its replication mechanisms, for testingneutralizing antibodies or antiviral agents, and likewise for developingbiological materials, diagnostic assays and vaccine preparations.Indeed, although the complete sequence of HCV has been available since1989 (Q. L. Choo et al., Science 244, 359 (1989)), the understanding ofthe life cycle and of the mode of replication of HCV has been hinderedby the lack of a suitable system of in vitro culture. Ito et al. (J.Gen. Virol. 77: 1043-1054 (1996)) have indeed confirmed the maintenanceof the replication of HCV in primary cultures of human hepatocytesobtained from patients carrying HCV and for whom the disease waschronically established, and suggested a passage of infection, butproblems relating to the propagation of the virus remain (impossibilityof long-term culture) and the system developed is limited by the need ofa supply of human liver and the laborious nature of the technique.Moreover, to date, there is no general consensus as to the tropism ofHCV, and not all the cell receptors for the virus have yet beenidentified.

Particles containing viral RNA that are very heterogeneous in terms ofdensity are found in the plasma of HCV-infected patients. Thisheterogeneity of density of the particles containing viral RNA isattributed to their association in variable proportion with lipoproteins(Thomsen et al., 1993, Med. Microbiol. Immunol. 182:639). In thedescription of the present patent application, the inventors have calledthese hybrid particles LVPs (lipo-viro-particles). The distribution ofeach of these forms along a density gradient varies from one patient toanother. Existing analyses of the low-density particles show densitiescovering those of LDLs (low density lipoproteins) and of VLDLs (very lowdensity lipoproteins).

The nature of the LVPs containing viral RNA is not at this timeprecisely known.

Patent application WO 01/09289 describes a method for culturing viruses,such as HCV, in vitro, from at least one fraction of LVPs obtained fromserum or plasma of an infected patient, using cells which have anendocytosis pathway relayed by at least one lipoprotein receptor andmodulated by an activating agent. This method endeavors especially topromote the entry of the fractions into the cells in culture. However,the replication of the virus remains limited and must therefore beoptimized.

Patent application WO 02/10353 describes complexes consisting of LVPsassociated with human immunoglobulins, having a density of less than orequal to 1.063 g/ml, and containing mainly HCV virus RNA, contrary toknown data (Hijikata et al., 1993, J. Virol., 1953-1958). In fact,Hijikata et al. showed that a high infectivity was found in chimpanzeesin the presence of particles having a density of less than 1.06 g/ml,such that there could not be human immunoglobulins, which are verydense, in this type of low-density particles. These complexes constitutea particular form of the HCV virus which is very infectious.

That patent application also describes a method for culturing the HCVvirus in vitro, from the LVP/immunoglobulin complexes, using cellscomprising at their surface at least one type of immunoglobulin Fcfragment receptor or one type of receptor having the ability to bindimmunoglobulins. Here again, this method promotes the entry of thecomplexes into the cells in culture, but it does not allow an abundantmultiplication of the virus.

The present inventors have now found a novel method for culturing theHCV virus in vitro, which makes it possible to solve the above drawback,namely it uses cells capable both of causing the entry of the particlescontaining the HCV-RNA and of improving the replication and theproduction.

In fact, the present inventors have demonstrated, surprisingly, that theparticles of HCV RNA, called LVPs:

-   -   are unit particles that are spherical overall; they are not        therefore agglomerates of virions bound to normal lipoproteins,        as had been suggested in the prior art,    -   contain apolipoproteins B and E and triglycerides: they        therefore have a lipoprotein structure, and in particular a        structure of the VLDL or chylomicron type, and    -   contain the capsid and the RNA of the virus: they therefore        differ from the normal lipoproteins that are found in humans and        constitute an unconventional form of the virus.

Since the LVPs are hybrid particles, i.e. lipoproteins containing viralcomponents, the replication can be unexpectedly improved using cellscapable of synthesizing the lipoproteins.

Thus, a subject of the present invention is a method for culturing theHCV virus in vitro, comprising the steps consisting in bringingparticles containing the RNA of the hepatitis C virus into contact withcells having the ability to synthesize and to secrete lipoproteins, in asuitable culture medium that promotes the synthesis and the secretion ofthe lipoproteins, and in harvesting the virus thus obtained.

The expression “particles containing the RNA of HCV” is intended to meanviral particles such as, in particular, in the form of anLVP/immunoglobulin complex or present in the serum or the plasma ofpatients detected to be HCV-positive and containing such particles, andany inoculant containing the viral RNA, whatever the mode of preparationof the viral RNA in said inoculant.

The LVP/immunoglobulin complexes and the method for purifying them froma sample of plasma or of serum of a patient infected with HCV havealready been described in patent application WO 02/10353.

However, that document neither described nor suggested the particularnature of these LVPs of a lipoprotein type, i.e. whether they possesstriglycerides, apolipoprotein B and, optionally, apolipoproteins E.

Apolipoprotein B is a human protein which is associated with theendoplasmic reticulum membrane and which initiates the assembly ofVLDLs, the introduction of triglyceride in the presence of microsomaltriglyceride transfer protein, and also the assembly of VLDLs in thelumen of the endoplasmic reticulum. This apolipoprotein is provided intwo forms, apo B 100 and apo B 48, and is synthesized in the liver andthe intestine.

Apolipoprotein E can be synthesized by many cells and in particular byhepatocytes. Moreover, it can be acquired by the VLDLs in thebloodstream.

The cells used in the method of the invention are all cells that havethe ability to synthesize and secrete lipoproteins.

These cells may be either primary cells or cell lines.

The term “cell line” refers to established lines that are immortalizedspontaneously or by manipulation. In practice, to carry out a viralculture of interest, it is necessary to have permissive cells that arereadily maintained in culture. The cell line is therefore preferably anestablished cell line or a cell line which results from immortalizationby various methods. This may be carried out (i) by the establishment ofa stable, established, continuous line by co-culturing permissive cellswith tumorized permissive cells of the same nature, which are capable ofmultiplying indefinitely and of ensuring the propagation of the viruswithin the culture, the viral inoculation taking place within theculture, (ii) using primary cells infected with the virus which are thencocultured with permissive tumorized cells which ensure the propagationof the virus within the culture of the cell line thus established, (iii)by viral infection of a cell line, for example an immortalized Blymphocyte line, for example with the Epstein-Barr virus, or else (iv)by modification of the telomerase activity.

The expression “cells having the ability to synthesize and secretelipoproteins used in the method of the invention” is intended to mean inparticular cells which have this ability spontaneously and cells whichhave acquired this ability after induction in a culture medium whichpromotes the differentiation or redifferentiation of the cells, or aftertransfection of genes for synthesis of the apolipoprotein apo B and ofthe microsomal triglyceride transfer protein MTP.

Examples of cells which have the ability to synthesize and secretelipoproteins that are suitable for the purposes of the inventioncomprise intestinal epithelial cells of the enterocyte type, brain cellsand liver cells.

According to one embodiment, the cells used are cells which haveacquired the ability to synthesize and secrete lipoproteins afterinduction in a medium that promotes the differentiation orredifferentiation of the cells.

Indeed, some cells, such as liver cells, have lost their ability toproduce lipoproteins, or other cells, such as intestinal epithelialcells, have an ability which can be improved. To overcome this drawback,it is possible to induce differentiation or redifferentiation of thesecells by bringing them into contact with a suitable medium that promotesthis differentiation or redifferentiation.

According to a particular embodiment, the cells that are suitable forthe purposes of the invention are intestinal epithelial cells, and inparticular Caco-2 cells (Van Greevenbroeck, M. M. J., et al., 2000,Atherosclerosis, 25-31).

In order to promote their ability to synthesize and secretelipoproteins, the intestinal epithelial cells, such as Caco-2, can bepre-cultured for 3 weeks with a DMEM medium supplemented with 10% offetal calf serum, in dishes coated with collagen or under semipermeablemembranes.

By way of examples of liver cells, mention may be made of hepatocytes(Moshage, H., et al., 1992, Journal of Hepatology, 15, 404413) andhepatocarcinomas such as Hep G2 cells (Gherardi, E., et al., 1992,Journal of Cell Science, 103, 531-539).

According to another particular embodiment, the cells used in the methodof the invention are hepatocarcinoma cells.

However, in this case, their ability to synthesize and secretelipoproteins can be promoted by bringing these cells into contact with amodified DMEM medium, i.e. containing at least one agent for inducinglipoprotein synthesis. These cells are preferably Hep G2 cells.

As an agent for inducing lipoprotein synthesis, mention may be made ofvarious lipids, such as fatty acids, preferably C₁₈ or C₂₀ fatty acids,for example oleate (Luchoomun, J., et al., 1999, The Journal ofBiological Chemistry, Vol 274(28), 19565-19572), and phospholipids suchas lysophosphatidylcholine (Zhou, Z., 1998, Biochimica et BiophysicaActa, 1391, 13-24).

The use of oleate in the hepatocarcinoma redifferentiation mediumconstitutes a particular embodiment of the invention.

According to one embodiment of the invention, the modified DMEM mediumconsists, besides of DMEM (Gibco BRL) and an agent for inducinglipoprotein synthesis, of 1% HEPES (Gibco), of 1% glutamine (Gibco), of0.25 mg/ml of gentamycin (Gibco), of 1.5% Ultroser-G (Gibco), of 5×10⁶ Mforskolin (ICN), of 1.6×10⁻⁷ M PMA (4-α-phorbol 12-myristatel13-acetate)(Sigma), of 5.6 IU/ml of retinol acetate (Sigma), of 0.5×10⁻³ M sodiumbutyrate (Sigma), of 10⁻² M niacidamide (ICN), of 2×10⁻⁶ g/ml ofpolybrene. (Sigma), of 2.9×10⁻⁸ M sodium selenite (ICN) and of 1×10⁻⁹ Mtriiodo-L-thyronine sodium salt (ICN).

Another type of cells that can be used in the method of the inventionconsist of the cells obtained after transfection of genes for synthesisof the apolipoproteins apo B and, optionally, apo E and of themicrosomal triglyceride transfer protein. By way of example of suchcells, mention may be made of transfected insect cell lines as describedby D. G. Gretch, et al., in The Journal of Biological Biochemistry,1998, Vol 271(15), 8682-8691.

The particles containing the HCV RNA, when they are brought into contactwith the cells that have the ability to synthesize and secretelipoproteins, enter into these cells either via the lipoproteinreceptors of said cells, of the LDL receptor type, as in the case of theLVPs for example, or by means of internalization by transfectionaccording to techniques known to those skilled in the art, as in thecase of viral RNA preparations, for example.

The culture medium used in the method of the invention is thus that itpromotes the synthesis and the secretion of lipoproteins, preferably ofVLDL or chylomicron type.

A suitable medium that is selected is a modified DMEM mediumsupplemented with agents that promote the metabolism of the cell inculture, and also with at least one agent for inducing lipoproteinsynthesis.

As agents that promote the metabolism of the cell, mention may be madeof dexamethasone and insulin.

A preferred modified DMEM medium is as defined above and supplementedwith dexamethasone and insulin.

As agents for inducing lipoprotein synthesis, mention may be made ofvarious lipids, such as fatty acids, preferably C₁₈ or C₂₀ fatty acids,for example oleate, phospholipids such as lysophosphatidylcholine, andalso 22- or 25-OH-cholesterol.

The use of oleate as agent for inducing lipoprotein synthesis,optionally in combination with 22- or 25-OH-cholesterol, constitutesanother particular embodiment of the invention.

The virus thus cultured by means of the method of the invention can beharvested by various methods such as centrifugation, for example on adensity gradient, and immunoprecipitation using anti-apo B and/oranti-apo E antibodies.

The invention also relates to a diagnostic composition comprising atleast the viral particles obtained according to the method of theinvention or a component thereof as source of antigen.

Those skilled in the art will readily determine the amount of viralparticles to be used according to the diagnostic technique used.

The invention also relates to a method for screening for and/orselecting at least one antiviral molecule, comprising the step ofbringing said antiviral molecule into contact in the culture mediumduring the method of culture of the invention.

The selection of the antiviral molecules is carried out by means oftechniques well known to those skilled in the art, such as the reductionin the number of intracellular viral RNAs and/or of infectious viralparticles secreted into the supernatant in the presence of varyingconcentrations of the various inhibitors.

These inhibitors may be nucleotide or nucleoside analogs, or inhibitorsof viral proteases or of other molecules, which interfere with thefunctions of the other viral proteins.

However, the invention also opens up other therapeutic perspective inthat it makes it possible to develop a therapeutic composition capableof qualitatively and/or quantitatively influencing the propagation andthe replication, in vivo, of HCV, which composition is characterized inthat it comprises, inter alia, an agent capable of modulating,repressing or inhibiting lipoprotein synthesis, such as, for example, aninhibitor of the microsomal triglyceride transfer protein.

The expression “agent capable of modulating, repressing or inhibitinglipoprotein synthesis” is intended to mean any molecule making itpossible, respectively, to control, decrease or suppress the propagationand the replication, in vivo, of HCV.

These agents can be selected by screening from a pool of moleculeshaving recognized pharmacological activity on lipid metabolisms.

By way of example of an agent capable of inhibiting lipoproteinsynthesis, mention may be made of the microsomal triglyceride transferprotein inhibitor.

The invention will be understood more fully from the following examplesgiven by way of nonlimiting illustration, and also from the attachedFIGS. 1 to 3, in which:

FIG. 1 represents a graph showing the influence of the cultureconditions (modified DMEM medium plus oleate compared with standardmedium) for the Hep G2 cells on the secretion of viral HCV particles,which secretion is evaluated by quantifying the viral RNA in the culturesupernatant as a function of time;

FIG. 2 represents a graph showing the influence of the addition ofoleate to the modified DMEM culture medium on the secretion of viralparticles, which secretion is evaluated by quantifying HCV RNA in theculture supernatant as a function of time, and

FIG. 3 represents a graph showing the HCV virus production by Caco-2cells, after differentiation for 3 weeks of culture, said productionbeing demonstrated by the number of viral RNAs in the culturesupernatant as a function of time.

EXAMPLE 1 Characterization of LVPs

1.1 Demonstration of the Overall Spherical Form of the LVPs

The LVPs of sera from patients detected as being positive for thehepatitis C virus are purified as described in patent application WO02/10353.

The low density (LDL) fraction, from which the purified LVPs had beenremoved, and also the purified LVPs, were diluted in PBS and werevisualized using an electron microscope (JEOL device, Centre Commund'Imagerie de Laennec, Lyon, France) after having floated drops of thesample on copper grids with a mesh size of 200, that were coated with aFormvar support film (Electron Microscopy Science, Pa.) for 3 min atambient temperature, stained for 3 min by floating on a 4% (mass/vol)phototungstic acid medium, buffered at pH 7.2 with NaOH, and then dried.

The LDL fraction consisted of particles having a homogeneous sphericalstructure, with a mean diameter of 25 nm, in accordance with normalLDLs.

On the other hand, the purified LVPs were unusually large sphericalstructures, with a mean diameter of 100 nm.

1.2 Assaying of the LVP Constituents

a) Determination of the Lipid Concentration of the LVPs

The total cholesterol, phospholipid and triglyceride concentrations weredetermined using cholesterol RTU, phospholipids enzymatic PAP 150 andtriglyceride enzymatic PAP 150 kits (bioMérieux, Marcy l'Etoile, France)according to the manufacturer's recommendations, and by establishingstandard curves.

b) Determination of the apo B Concentration of the LVPs

The concentration of apolipoprotein apo B in the purified LVPs wasdetermined by means of an ELISA assay. To do this, 96-well,flat-bottomed ELISA plates (Maxisorb; Nunc) were coated with 100 μl ofmonoclonal anti-human anti-apo B antibody (5 μg/ml; clone 1609;Biodesign, Saco, Me.) in PBS (phosphate buffer saline solution). Theplates were left to stand overnight at 4° C., and then the reaction wasblocked with 2% of BSA (bovine serum albumin).

The samples were first of all incubated for 30 min at ambienttemperature in a mixture of PBS-0.2% BSA supplemented with 10 μg ofhuman IgG/ml, and were then distributed onto the plates in a proportionof 100 μl/well.

After incubation for 2 h at 37° C. and washing with a PBS-0.05% Tween 20medium, goat anti-human apo B antibodies conjugated to peroxidase (1.6μg/ml; Biodesign), in a PBS-0.2% BSA mixture, were added in a proportionof 100 μl/well, and the plates were left to incubate for 90 min at 37°C.

The plates were washed and the o-phenylenediamine substrate (Sigma) wasadded in a proportion of 150 μl/well. The reaction was allowed todevelop for 10 min and the plates were read at 490 nm.

c) Results

The results, in terms of triglyceride/cholesterol (TG/Chol) andtriglyceride/apo B (TG/apo B) ratio, are given in the table below. Byway of comparison, this table also contains these same ratios for thenormal lipoproteins of the patient, i.e. the lipoproteins obtained afterextraction of the LVPs. TABLE Normal lipoproteins Purified LVPs Fractiondensity TG/Chol TG/apo B TG/Chol TG/apo B <1.006 3.4 ± 1.7    15 ±1.3^(a) 3.1 ± 1.6  160 ± 47^(a) 1.025-1.055  0.45 ± 0.2^(b) 0.8 ±0.2^(a) 2.8 ± 1.9^(b)  26 ± 20^(a)^(a)p ≦ 0.04^(b)p ≦ 0.01

The LVPs, which are found in the two fractions of different density,namely low and very low densities, contain more triglyceride permolecule of apo B than the normal lipoproteins of the same fractions.

These data confirm that:

-   the LVPs indeed contain lipids,-   the lipid concentration of the LVPs is different from those of the    normal lipoproteins; this therefore excludes any contamination, and-   the LVPs contain apo B.

1.3 Presence of Apolipoproteins B and E

The presence of apo B and of apo E was demonstrated by preventing theentry of the LVPs into PLC cells after blocking the sites of binding tothe apo B and apo E receptors of said cells as follows:

5×10⁵ PLC/PFR/5 human hepatoma cell line cells (ATCC CRL 8024)(Alexander cells) (human hepatoma) were cultured in 96-well plates(Maxisorb, Nunc) for 24 h with a DMEM culture medium (Gibco, BRL)supplemented with 10% of fetal calf serum (Biowhittaker, Emerainville,France), 2 mM of HEPES (Gibco/BRL), 1% of nonessential amino acids(Gibco/BRL) and 50 IU of penicillin/streptamycin (Gibco/BRL)/ml at 37°C.

Firstly, the apo B receptor-binding sites were blocked with monoclonalantibodies directed against the apo B receptor-binding site (4G3 and5^(E)11; Ottawa Heart Institute Research Corporation, Ottawa, OntarioCanada) and, secondly, the apo E receptor-binding sites were blockedwith monoclonal antibodies 1D7 (Ottawa Heart Institute ResearchCorporation). The PLC cells were washed three times with PBS and wereincubated for 3 h with purified LVPs. The cells were washed and werethen harvested in 350 μl of lysis buffer from the Rneasy kit (Qiagen),and the RNA was extracted as indicated above.

The blocking of the recognition of the LVPs by blocking the apo B andapo E recognition sites indicates that they contain apolipoproteins anddemonstrates the lipoprotein structure of the LVPs.

1.4 Presence of the Capsid Consisting of the Core Protein and of the RNAof the Virus, in the LVPs

The presence of the capsid was demonstrated by delipidating the purifiedLVPs in the following way: the LVPs were incubated for 30 min, whilegently stirring, in a solution of 85% of ether-15% of butanol. Thepresence of the capsid was visualized using an electron microscope (JEOLdevice, Centre Commun d'Imagerie de Laennec, Lyon, France) as indicatedin point 1.1 above.

The presence of the HCV virus core protein was confirmed by Westernblotting by bringing the LVPs that had been delipidated as describedabove into contact with anti-HCV core protein monoclonal antibodies(19D9D6; Jolivet-Reynaud, C. P., et al., 1998, J. Med. Virol., 56,300-309) and 10 nm-gold labeled secondary antibodies, and visualizingusing the grids indicated above, by immunodetection after negativestaining by floating on 3% uranyl acetate.

Finally, the presence of the virus RNA was demonstrated by extraction ofthe purified and delipidated LVPs using a QIAamp kit (Qiagen S. A.Courtaboeuf, France).

Example 2 Influence of the Culture Conditions on the Production of HCVVirus by Hep G2 Cells

Hep G2 cells were first of all cultured either in a medium consisting ofDMEM and of 10% fetal calf serum (standard medium), as in patentapplication WO 01/09289, or in a modified DMEM medium, i.e. containingDMEM supplemented with 1% HEPES, 1% glutamine, 0.25 mg/ml of gentamycin,1.5% Ultroser-G, 5×10⁶ M forskolin, 1.6×10⁻⁷ MA, 5.6 IU/ml of retinolacetate, 0.5×10⁻³ M sodium butyrate, 10⁻² M niacidamide, 2×10⁻⁶ g/ml ofpolybrene, 2.9×10⁻⁸ M sodium selenite and 1×10⁻⁹ M triiodo-L-thyroninesodium salt.

Falcon 24-well culture plates were then seeded in a proportion of 150000 cells per well with standard medium or modified DMEM medium, andleft in culture for 24 h.

The medium was removed by suction and the cells were incubated in thepresence of the virus, in a proportion of 500 000 HCV RNAs per well, for6 h using DMEM supplemented with 0.2% of BSA.

The cells were then washed with PBS and were cultured either in standardmedium, or in modified medium, as indicated above, but also supplementedwith hexamethasone and insulin and a mixture of oleate and BSA in aproportion of 0.15 mM of oleate.

All the cultures were effected in a 5% CO₂ atmosphere and at 37° C.

A sample of supernatant was then taken from the wells (in triplicate)and the HCV RNA was quantified by RT-PCR according to the protocoldescribed by Komurian-Pradel, F. in J. Virol. Methods, 2001, 95,111-119.

The results, copies of RNA/ml of supernatant as a function of time, aregiven in FIG. 1, in which the diamonds represent the use of the standardmedium and the squares the use of the medium for promoting the synthesisand secretion of lipoproteins.

This figure demonstrates that the replication of the virus and thesecretion thereof are improved by using cells having the ability tosynthesize and secrete lipoproteins and by placing them under favorableconditions.

Example 3 Importance of the Addition of Lipids to the Medium forPromoting the Synthesis and Secretion of Lipoproteins

Hep G2 cells were first of all cultured for 24 h in a modified medium asdescribed in example 2 above, and were added to the wells of the Falcon24-well culture plates in a proportion of 150 000 cells per well.

The cells were then incubated in the presence of the virus, in aproportion of 400 000 HCV RNAs per well, for 6 h in a DMEM mediumsupplemented with 0.2% of BSA, alone or with 0.1 μM of25-OH-cholesterol.

The cells were then washed as indicated in example 2 above, and werecultured in modified medium.

Three days after the inoculation of the cells, an oleate-BSA mixturewith 0.15 mM of oleate was added.

The supernatant was collected and the HCV RNA was quantified by RT-PCR.

The results, demonstrating the importance of the use of oleate, aregiven in FIG. 2 in which the diamonds represent the use of the modifiedmedium alone and the squares the use of the modified medium supplementedwith oleate and with 25-OH-cholesterol.

Example 4 Culturing of the Virus Using Caco-2 Cells

Caco-2 cells are differentiated on semipermeable membranes (Transwell,Costar) contained in 24-well plates, for 3 weeks in standard medium(DMEM supplemented with 10% of fetal calf serum).

The cells thus prepared were incubated with the viral particles in aproportion of 200 000 HCV RNAs/well, for 6 h.

The cells were washed and were cultured in DMEM medium supplemented with10% of fetal calf serum and with oleate-taurocholate containing 0.15 mMof oleate.

The supernatant above the insert was taken, and the viral RNA wasquantified by RT-PCR.

The results are given in FIG. 3, which demonstrates the culturing of theHCV virus by means of intestinal epithelial cells in a medium forpromoting the synthesis and secretion of lipoproteins.

1. A method for culturing the hepatitis C virus in vitro, characterizedin that it comprises the steps consisting in bringing particlescontaining the RNA of the hepatitis C virus into contact with cellshaving the ability to synthesize and to secrete lipoproteins, in asuitable culture medium that promotes the synthesis and the secretion ofthe lipoproteins, and in harvesting the virus thus obtained.
 2. Themethod as claimed in claim 1, characterized in that the particlescontaining the RNA of the hepatitis C virus are LVP lipo-viro-particles.3. The method as claimed in claim 1, characterized in that the cellsthat have the ability to synthesize and secrete lipoproteins are chosenfrom cells that spontaneously have this ability and cells that haveacquired this ability after induction in a culture medium that promotesthe differentiation or redifferentiation of the cells or aftertransfection of genes for synthesizing apolipoprotein B and themicrosomal triglyceride transfer protein.
 4. The method as claimed inclaim 3, characterized in that the cells used are cells which haveacquired the ability to synthesize and secrete lipoproteins afterinduction in a medium that promotes the differentiation orredifferentiation of the cells.
 5. The method as claimed in claim 1,characterized in that said cells are intestinal epithelial cells.
 6. Themethod as claimed in claim 5, characterized in that said cells areCaco-2 cells.
 7. The method as claimed in claim 4, characterized in thatthe cells are pre-treated for 3 weeks with a DMEM medium supplementedwith 10% of fetal calf serum.
 8. The method as claimed in claim 1,characterized in that said cells are hepatocarcinoma cells.
 9. Themethod as claimed in claim 8, characterized in that the hepatocarcinomacells are Hep G2 cells
 10. The method as claimed in claim 8,characterized in that said cells are brought into contact beforehandwith a modified DMEM medium containing an agent for inducing lipoproteinsynthesis.
 11. The method as claimed in claim 10, characterized in thatthe agent for inducing lipoprotein synthesis is oleate.
 12. The methodas claimed in claim 1, characterized in that the medium that promotesthe synthesis and secretion of lipoproteins is a medium derived from themodified DMEM medium, supplemented with agents for promoting themetabolism of the culture cell, and also with at least one agent forinducing lipoprotein synthesis.
 13. The method as claimed in claim 12,characterized in that said medium comprises oleate as agent for inducinglipoprotein synthesis.
 14. The method as claimed in claim 13,characterized in that said medium also contains 22- or 25-OH-cholesterolas another agent for inducing lipoprotein synthesis.
 15. A method forscreening for and/or selecting at least one antiviral molecule,characterized in that it comprises the step of bringing said antiviralmolecule into contact in the culture medium during the method of cultureas claimed in claim
 1. 16. A therapeutic composition capable ofqualitatively and/or quantitatively influencing the propagation and thereplication, in vivo, of HCV, characterized in that it comprises, interalia, an agent capable of modulating, repressing or inhibitinglipoprotein synthesis.